By Michiel Mueller
This booklet offers a entire account of the speculation of photograph formation in a confocal fluorescence microscope in addition to a pragmatic instruction to the operation of the software, its obstacles, and the translation of confocal microscopy facts. The appendices supply a brief connection with optical conception, microscopy-related formulation and definitions, and Fourier theory.
- Symbols and abbreviations
- Confocal fluorescence microscopy
- sensible limits
- Miscellaneous topics
- Appendix A: components of optical theory
- Appendix B: formulation, family and definitions
- Appendix C: Fourier theory
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Additional info for Introduction to Confocal Fluorescence Microscopy, Second Edition
The advantage of this arrangement is that it combines confocal microscopy with cooled CCD cameras. These cameras have the useful properties of high sensitivity and remarkable linearity in combination with a high dynamic range. In the above description of the working principle of the bilateral scanning technique, it is explicitly noted that the light source may be either a pinhole, as discussed so far, or some other pattern. Another useful illumination pattern is the line cursor. In this case, the illumination does not originate from a single point (pinhole) but from a line or slit.
Then the confocal PSF is simply the square of the PSF of excitation, PSFconf = |PSFexc |2 . 17) Clearly, in this case the Rayleigh criterion cannot be used directly to define the resolution in a confocal microscope since it is related to the position of the first minimum in the distribution. The position of this minimum does not change upon squaring the distribution. To circumvent this problem while retaining the convenience of the analytic expression of Eq. 15), one generally uses the FWHM of the distribution.
In addition to the induced focal shift, the PSF broadens significantly, especially in the axial direction. 18 shows an accumulation of the results shown in Fig. 17 for the confocal PSF. Depicted are the magnitude of the induced focal shift, the FWHM of the on-axis distribution, and the amplitude of the maximum of the distribution, all as a function of the NFP. 17 The influence of the refractive index mismatch as a function of the focusing depth on the excitation and confocal PSF (see text for details).
Introduction to Confocal Fluorescence Microscopy, Second Edition by Michiel Mueller